A common and recurring problem in both the research laboratory and the clinical testing laboratory is the maintenance of the test specimen in a manner which prevents degradation, alteration, or destruction of essential test materials. The need for preservation of the test specimen is particularly great when the sample is a body fluid taken from an animal or human patient and from which an analytical determination and measurement must be made in order to evaluate and understand the subject's medical status. Typically, the first question is which fraction of the body fluid sample is of interest for analytical or evaluation purposes? Thus, either the cellular fraction comprising whole cells, cell fragments, and the like is of primary interest; or the non-cellular solubilized fraction containing all the dissolved chemical constituents and components presents the specific entities to be detected, identified, and analyzed. The process of separating the cellular components from the solubilized liquid fraction is easily achieved using the conventional techniques of centrifugation, filtration, precipitation, sedimentation, and the like. In addition, a number of other classical means have been developed for removal of interfering substances from the sample in order to assure specificity of an analytical method. Among the conventional approaches typically used are dialysis, a process by which constituents of low molecular weight are separated across a semi-permeable membrane from compounds having a high molecular weight; column chromatography, which remove compounds that would adversely affect test reactions using gel filters, ion exchangers, or chemical resins; as well as other separation systems in which a layer of material separates or excludes compounds of low molecular weight from interference of large molecular weight.
It has long been recognized that the body fluid sample must be maintained and preserved during the manipulations and preparations preceeding analysis as a test specimen. For these reasons, a number of different compositions have been developed for maintaining the stability of the cellular fraction or the non-cellular components of a test sample during the preparatory stages. Merely illustrating and exemplifying such stabilizing compositions are the following: The use of two protease inhibitors, anastatin and leupeptin, in combination with EDTA for stabilizing peptides in whole blood, serum, or plasma samples [U.S. Pat. No. 5,541,116]; the use of a water-soluble phosphate such as ATP and a chelating agent for the preservation of whole cells or cellular components [Publication EP 431385-A]; the use of an acid, anti-bacterial drug, and fluorine compound in combination for stabilization of cells in urine [Japanese Patent Publication 05249104-A]; the use of an aqueous solution of ethanol, aliphatic diol and polyethylene glycol for preserving cell or blood fluid components [Japanese Patent Publication 03295465-A]; a reagent composition for biological assays which contains a reducible water-soluble trivalent cobalt complex, metallisable dye, and water-soluble polymer [U.S. Pat. No. 5,171,669]; a stability control solution for determination of urobilinogen in urine samples [U.S. Pat. Nos. 4,677,075 and 4,703,013]; the use of an aqueous solution containing phosphate buffer, albumin, glycine, and cysteine for stabilizing dehydrogenases [German Patent Publication DE2629808-A]; a stabilizing composition comprising a buffer, alanine and mannitol for stabilization of freeze-dried protein compositions [Publication EP682944-A1]; the use of cationic poly-electrolyte and cyclic polyiol in aqueous solutions to stabilize proteins against denaturation on drying [U.S. Pat. No. 5,240,843]. In addition, a number of stabilizing preparations have been commercially manufactured and sold, a notable example being the COMPLETE protease inhibitor cocktail tablets for the inhibition of proteases during extractions from animal and plant tissues.
Despite the development and commercial availability of stabilizing preparations and compositions, the overwhelming majority of these are quite limited as to their usefulness and efficacy; and do not lend themselves without major modification and alterations to specific clinical problems or a broad variety of different clinical and analytical settings. A particular example will illustrate the deficiency. The example involves the treatment of human bladder cancer using the immunotherapeutic agent Bacillis Calmette-Guerin (BCG) as an intravesical agent.
The BCG Example
Since its isolation from Mycobacterium bovis, a form of cow tuberculosis, in 1921 by co-workers Calmette and Gue rin at the Pasteur Institute, BCG has found widespread medicinal use. As an anti-tuberculosis vaccine, it has been administered successfully to over 2.5 billion people worldwide, conferring a protection rate of between 70% to 80% [Lvelmo, F., Am. Rev. ResPir. Dis. 125: 70 (1982)]. Its potential use as an anti-cancer agent was suggested by the work on bacterial toxins by Coley in the late 1890s, as well as by the observation of Perle in 1929 that patients with tuberculosis had a lower incidence of cancer [Nauts et al., Acta Medica Scand. 276: 5 (1953); Perle, R., Am. J. Hygiene 9: 97 (1929)]. The first use of BCG against human cancer was reported in 1935, and its immunostimulatory properties were realized later that decade [Holmgnen, I., Schwerz. Med. Wochenschr 65: 1203 (1935); Van der Meidjer et al., Proa. Clin. Biol. Res. Bio.: 11 (1959)]. However, it was not until the late 1950s and 1960s that experimental and clinical studies generated enthusiasm for its use against cancer.
BCG's potential use in bladder cancer was encouraged by the work of Coe and Feldman demonstrating the immunocompetence of the bladder; and by the observations of Zbar and his colleagues that close contact between BCG and tumors was required for efficacy [Coe, J. E. and J. D. Feldman, Immunol. 10: 27 (1966); Zbar et al., J. NatI. Cancer Inst. 49: 119 (1972)]. The 1980 controlled study by the Southwest Oncology Group confirming BCG's efficacy against superficial bladder cancer ushered forth the modern era of BCC immunotherapy for bladder cancer [Lamm et al., J. Urol. 124: 38 (1980)]. Not only was BCG established as an effective intravesical agent for bladder cancer, but it also came to be regarded, in many cases, as the agent of choice.
BCG has been used successfully in superficial transitional cell carcinoma (TCC) of the bladder as a prophylactic agent to reduce tumor recurrence and as a therapeutic agent to treat unresectable residual disease or carcinoma in situ (CIS). Its superiority to transurethral resection of bladder tumors in reducing tumor recurrence has been documented by several independent studies. With the possible exception of mitomycin C usage, BCG has also proved to be more effective in reducing tumor recurrence than all other forms of conventional intravesical therapy. Possibly because of its intrinsic tendency to remain on the most accessible surface of the bladder, CIS particularly has proved to be responsive to BCG therapy with complete response rates ranging between 70% to 80%. This is especially noteworthy as CIS is not accessible to local surgical management and carries an 80% chance of disease progression if left untreated. Compared with CIS, the response rate of more bulky residual superficial bladder cancer to BCG is lower but still is a respectable 50% to 60% rate.
The successful application of BCG immunotherapy to bladder cancer proceeds through at least two phases: (1) an initiation phase and (2) an effector phase. During the initiation phase BCG attaches to and is retained by the bladder in an immunologically active form; and it is now clear that a significant portion of BCG attachment to the bladder is fibronectin dependent. After fibronectin attachment, BCG is phagocytosed by macrophages and bladder epithelial cells. The latter process is mediated by integrin receptors. Also, after ingesting BCG, bladder epithelial cells have the capacity to present BCG derived antigens on their cell surface for immune recognition.
Analysis of the effector phase of BCG therapy is complicated by the ability of BCG to activate multiple cellular compartments including macrophages, natural killer (NK) cells, B cells, and various T cells (helper, cytotoxic, and gamma-delta). Clinically, this is manifested by marked pyuria soon after BCG installation that reaches its height during the last two of the usual six treatments. During this same period, potent biologic proteins termed cytokines are measurable in the urine--with interleukin 2 (IL-2), tumor necrosis factor-alpha (TNF-.alpha.), and interferon gamma (IFN-.gamma.) peaking during later instillations. Progressive resolution of positive urinary cytologies, indicative of tumor presence, parallels these late cytokine responses. A general review of these events and the future prospects for BCG therapy is provided by O'Donnell, M. A. and W. C. DeWolf, Surgical Oncology Clinics of North America 4: 189 (1995) and the references cited therein.
Substantial research interest and clinical experimentation has centered on the accurate detection and quantitation of various cytokines in the urine of patients with superficial bladder tumors who have undergone treatment with intravesical BCG. In the main, two goals are sought. The first broad purpose is to understand and describe the presence of these urinary cytokines as consequences of antitumor activity of intravesical BCG treatments. Merely representative of such research investigations are the following scientific reports: Haaff et al., J. Urol. 136: 970 (1986); Bohle et al., J Urol. 144: 59 (1990); Balbay et al., Urology 43: 187 (1994); Prescott et al., J. Urol. 144: 1248 (1990); Jackson et al., J. Urol. 148: 1583 (1992) and the references cited within these individual publications. The second broadly stated goal is the potential use of these urinary cytokines as in-vivo prognosticators of intravesical BCG treatment efficacy. This area of research is representated by: Thalmann et al., J. Urol. 155: 34A (1996); O'Donnell et al., J. Urol. 155: 1030A (1996); DeReijke et al., J. Urol. 155: 477 (1996). and the references cited within each of these individual publications.
A major problem, however, in all the reported investigations is that the cytokines in the urine samples are highly unstable. Many interleukins were found degraded and/or lost in urine samples held at 4.degree. C. and 20.degree. C., while all cytokines were found to be destroyed at 37.degree. C. Moreover, other individual cytokines such as interferon-gamma could only be detected in immediately dialyzed urine; no freezing, preparation, or known stabilizing agent served adequately to prevent degradation and destruction of these proteins. In fact, the only reliable means of stabilizing and maintaining the protein integrity of the individual cytokines was immediate dialysis of the samples directly after the urine was voided by the living patient. Insofar as is known to date, no preservative, stabilizer, or maintenance composition has been effective to prevent the degradation and destruction of cytokines present in a body fluid sample intended for evaluation as a clinical or analytical test specimen.
Accordingly, from the specific BCG example described as well as from the general history of stabilizing or maintenance preparations previously known and conventionally used in this field, there remains a long standing need for a broadly effective stabilizing medium which will truly maintain and preserve the integrity of solubilized proteins such as cytokines from deterioration, alteration and destruction in a test specimen. Should such a stabilizing formula be developed, this preparation and formulation would be seen as a major advance in this technical area; and would provide substantive advantages and benefits to research investigators and clinical practitioners who routinely evaluate body fluid samples taken ex-vivo from living humans and animal patients.